Like many transmembrane proteins, determination of the structure of P-gp by X-ray crystallography has proven very difficult. This stems from the problems encountered forming sufficient-quality crystals that maintain the native physiochemical environments for the different parts of the protein, and thus the native conformations. After many years of endeavor, a structure of the closely-related mouse P-gp protein has become available this last year. However, many questions remain as to how close the crystal structure relates to the protein in vivo, and how the conformation changes as part of the transport function. To address these questions, we are striving to integrate all available crystallographic and indirect experimental data with physiochemically-based mathematical methods to produce advanced models of the structures. Fortunately, over three decades of study has provided a wealth of information about P-gp from which we can gleam structural information. In addition to mouse P-gp, crystallographic structures are available from homologous proteins: especially bacterial Sav1866 and the MsbA lipid flippase. Examples of useful indirect experimental data include the effects of site-directed mutagenesis, naturally occurring polymorphisms, and residue cross-linking. Examples of theoretical, physiochemically-based methods include examining the patterns of residue conservation and polarity/hydrophobicity within the family of closely related MDR proteins and the superfamily of ABC transporters. This information helps predict which residues are exposed to the core and headgroup layers of the membrane, which residues line the pore, and which are at the interfaces of the two transmembrane domains. To this end, we are developing a grand sequence alignment of homologous families and the superfamily. The results of this will also enable the determination of patterns of correlated mutations, which help identify groups of residues that are proximal in the 3-dimensional structure of the protein. We have used our 3-D structural modelling of human P-gp to determine where to put electron paramagentic probes to experimentally determine different conformational states over the functional cycle of the protein. Additionally, we will use computational methods with our P-gp models to select nucleotide analogs and labeling agents to interact with and further elucidate the structure and functional mechanisms. Most recently, we have use the models to explain the experimentally-determined binding-effects of 5'-fluorosulfonylbenzonyl-5'-adenosine (FSBA), an ATP analogue, on the functional mechanisms of P-gp. We have also used computational methods to better account for the membrane environment on the protein. This last year, the models were used to design and interpret experiments that revealed the functional flexibility of the P-gp binding pocket. That is, mutation of known substrate-binding residues identified alternative, sub-pockets that allow for functional transport.